This invention relates to the field of analytical chemistry, more specifically to the field of spectrophotometric analysis of the transition metal vanadium and its oxidation state in pharmaceutical preparations and biological samples.
Introduction
Vanadium is a trace metal with remarkable properties. It believed to be an essential nutrient for many species, including man. Vanadium is a pervasive element of biological systems, being widely distributed across the food supply. At higher intakes, it accumulates in body tissues such as liver, kidney and bone. Vanadium has been shown to be useful as a therapeutic agent; for chemoprotection against cancers in animals (Bishayee and Chatterjee (1995); Djordjevic (1995)) as well as to alleviate the symptoms of diabetes by acting as an insulin mimetic (Tolman et al. (1979); Heyliger et al. (1985); Meyerovitch et al. (1987)).
The oxidation state of vanadium influences the biodistribution. In fact, incorporated vanadium appeared to be exclusively in the vanadyl (4+ oxidation state) form, and not the vanadate (5+ state). On examination of vanadyl ion in vitro, Sakurai et al. (1995) Biochem Biophys Res Commun 206(1):133-137, found that incubation of DNA with vanadyl ion and hydrogen peroxide (H2O2) led to intense DNA cleavage, and proposed that the mechanism for vanadium-dependent toxicity as well as its antineoplastic action was due to DNA cleavage by hydroxyl radicals. The data suggests that vanadyl compounds are less toxic than vanadate. However, vanadium is highly susceptible to oxidation under ordinary conditions. Crans et al. (1995) studied the stability of vanadium compounds. Several compounds including those currently favoured as insulin-mimetic agents were unstable in distilled water at pH 7. Even well characterized vanadium compounds were surprisingly labile.
On the basis of a number of studies (for example, see Erdmann et al. (1984); Nakai et al. (1995)), the vanadyl state has been proposed to be the active form of vanadium for insulin mimetic action, and is responsible for the positive actions of vanadium in vivo. At the same time, because vanadium tends to oxidize very easily, making stable and safe preparations of vanadium therapeutics is a challenge. It is therefore important to determine the oxidative state of vanadium before and after pharmaceutical administration.
Relevant Literature
Various kinds of samples have been analyzed for trace amounts of vanadium as a biological nutrient (Hurley in Trace Element Analytical Chemistry in Medicine and Biology, ed. Bratter et al., Berlin, 1984, vol. 3, p. 375), epidemiological preventive (Mracova et al., Science Total Environ, 1993, part 1, E16/633), pollutant (Langard S., and Norseth, T., in Handbook on the Toxicology of Metals, ed. Friberg, L, Nordberg, G F, and Vouk, V B., Elsevier, Amsterdam, 1986), and occupational hazard (Occupational Diseasesxe2x80x94A guide to their recognition. ed. Key et al., U.S. Department of Health, Education and Welfare, U.S. Government Printing Office, Washington D.C., June 1977).
Spectroscopic studies of oxovanadium (IV) complexes of biguanide, dibiguinide and 0-methyl-1-amidinourea were performed by Syamal (1983) Ind. J. Pure and Applied Physics 21:130-132. ESR, IR and electronic spectra were recorded. Indirect determination of vanadium may be performed by atomic absorption spectrometry (Chakraborty et al. (1989)).
In Keller et al. (1991) complexes of vanadyl, were reported to be formed with the trihydroxamic acid deferoxamine (H3DF+) with one complex exhibiting a characteristic reddish-violet color with a major absorbance peak at 386 nm and a smaller peak at 520 nm. In Rodriguez et al. (1994), oxyvanadium was reacted with molybdic acid in the presence of phosphate to form molybdivanadophosphoric acid absorbing at 385 nm and yellow in colour. In Bajeva et al. vanadate was reacted with N-m-tolyl-p-methoxy benzohydroxamic acid to form a 1:2 (metal to ligand) complex containing a basic Vxe2x95x90O group and an acidic Vxe2x80x94OH group, which formed addition compounds with thiocyanate to give a hyper- and bathochromic effect in chloroform. On the basis of this bathochromic effect of thiocyanate the spectrophotometric determination of vanadate was possible, in that the blue colored complex of vanadate could be extracted in chloroform, and had an absorption maxima at 580 nm.
Elvingson et al. (1996) speciated vanadium maltol in saline using NMR, ESR and potentiometric techniques. Takaya and Sawatari (1994) Ind. Health 32:165-178, speciated vanadium using ion-exchange chromatography and ICP-AES.
Ahmed and Banerjee provided a method for the spectrophotometric determination of vanadate using 5,7-dibromo-8-hydroxyquinoline (DBHQ) in slightly acidic solution. They attempted to distinguish between vanadyl and vanadate by masking vanadyl using tartrate and measuring absorbance at 400 nm in ethanol.
Methods are provided for the simultaneous quantitation of the (+IV) vanadyl and (+V) vanadate oxidation forms of vanadium. A sample is combined with a colorimetric substrate that forms a complex with the vanadium species, and which complexes differentially absorb light when formed with vanadyl or with vanadate. The buffers in the assay are chosen to minimize oxidation of the vanadium during the assay procedure. The methods are particularly useful for monitoring the oxidation of vanadyl to vanadate during manufacturing, pharmaceutical administration, etc. Oxidation may be determined at a single point, or over a time course, e.g. under conditions suspected of causing vanadyl oxidation. In a preferred embodiment, the colorimetric substrate is 5,7-dibromo-8-quinolinol (broxyquinoline), which provides for differential absorption of vanadyl at 400 nm and 525 nm.